ABCA1-mediated biogenesis of nascent HDL particles

描述（由申请人提供）：高密度脂蛋白胆固醇（HDL-c）是一种特别有前途的治疗性介入治疗冠心病（CHD）的候选者。尽管高密度脂蛋白c的绝对水平与冠心病风险呈负相关，但越来越多的证据表明，从外周组织到肝脏的高密度脂蛋白颗粒中的胆固醇通量——即胆固醇逆向转运（RCT）——是一个更重要的动脉粥样硬化保护因素。最近的研究表明，不同的HDL颗粒种类对RCT的刺激程度不同。粒子非均质性的起源尚不清楚。在新生的高密度脂蛋白中，一些粒子的形态形成已经很明显。新生HDL由细胞脂质和细胞外载脂蛋白AI （apoAI）通过atp结合盒转运蛋白A1 （ABCA1）介导的过程组装而成。在具体目标1中，我们提出验证ABCA1在质膜不同微环境中的定位是新生HDL颗粒异质性的原因。质膜的脂质组成和ABCA1在不同质膜结构域的假定定位将被操纵，以确定这些操作对新生HDL群体的影响。ApoAI结合保护ABCA1免受降解，并在前馈调节回路中促进新生HDL颗粒的形成。在Specific Aim 2中，我们提出了两种主要方法来鉴定ABCA1上假定的apoAI结合位点。在一种方法中，ABCA1和apoAI将交联，然后ABCA1-apoAI复合物将使用质谱分析。在第二种方法中，计算选择的候选ABCA1区域将被化学合成，并使用表面等离子体共振测试与apoAI的结合。用这两种方法鉴定的ABCA1区域将通过诱变分析和结合完成试验进行验证。从这个项目中获得的知识将有助于设计新的疗法来刺激随机对照试验，并最大限度地产生最能保护动脉粥样硬化的高密度脂蛋白。

DESCRIPTION (provided by applicant): High-density lipoprotein cholesterol (HDL-c) is an especially promising candidate for therapeutic intervention against coronary heart disease (CHD). Although, absolute levels of HDL-c correlate inversely with the CHD risk, there is growing evidence that cholesterol flux in HDL particles from peripheral tissues to the liver - i.e., reverse cholesterol transport (RCT) - is a more important atheroprotective factor. Recent research shows that different HDL particle species stimulate RCT to varying extents. The origins of particle heterogeneity are unclear. Some particle speciation is already evident in nascent HDL. Nascent HDL is assembled from cellular lipids and extracellular apolipoprotein AI (apoAI) through a process mediated by ATP-binding cassette transporter A1 (ABCA1). In Specific Aim 1, we propose to test the hypothesis that localization of ABCA1 in different microenvironments of the plasma membrane is responsible for nascent HDL particle heterogeneity. Lipid composition of the plasma membrane and putative localization of ABCA1 to different plasma membrane domains will be manipulated to determine what effects these manipulations exert on the nascent HDL population. ApoAI binding protects ABCA1 from degradation and promotes nascent HDL particle formation in a feed-forward regulatory loop. In Specific Aim 2, we propose two primary approaches to identify putative apoAI binding sites on ABCA1. In one approach, ABCA1 and apoAI will be cross-linked and then ABCA1-apoAI complexes will be analyzed using mass spectrometry. In the second approach, computationally selected candidate ABCA1 regions will be chemically synthesized and tested for binding to apoAI using surface plasmon resonance. The regions of ABCA1 identified with the two approaches will be validated using mutagenic analyses and binding completion assays. The knowledge gained from this project will aid in design of novel therapies to stimulate RCT and maximize production of the most atheroprotective HDL species.